Elimination of immune responses to viral vectors

ABSTRACT

The present invention relates to the use of immunogenic peptides comprising a T-cell epitope derived from a viral vector antigen and a redox motif such as C-(X)2-[CST] or [CST]-(X)2-C in the prevention and/or suppression of immune responses to viral vectors and in the manufacture of medicaments therefore.

This application is a divisional of U.S. application Ser. No. 12/735,754(published as US 2011-0111502 A1, issued as U.S. Pat. No. 9,044,507 onJun. 2, 2015), filed Aug. 13, 2010, which is a U.S. national phase ofInternational Application No. PCT/EP2009/051803, filed 16 Feb. 2009,which designated the U.S. and claims priority to European ApplicationNo. 08447008.7, filed 14 Feb. 2008 and U.S. application Ser. No.61/035,826, filed 12 Mar. 2008, the entire contents of each of which arehereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to immunogenic peptides and their use inpreventing and/or suppressing immune responses to viral vectors such asused in gene therapy and in gene vaccination.

BACKGROUND OF THE INVENTION

Viruses offer a great potential as source of vectors for gene therapyand for gene vaccination. Several viruses are currently used for genetherapy, both experimental and in man, including RNA viruses(gamma-retroviruses and lentiviruses) and DNA viruses (adenoviruses,adeno-associated viruses, herpes viruses and poxviruses). The choice ofa virus vector is dictated by several factors, such as the time duringwhich transgene expression is required, the target cells that have to betransduced, whether the target cell is dividing or not, the risk relatedto multi-insertional events and the risk of inducing a vector-orientatedimmune response. For a recent review see, e.g., Flotte (2007), J. Cell.Physiol. 213, 301-305.

Gene therapy is now being considered for the treatment of an increasingnumber of diseases. These include: (1) autosomal recessive single genedisorders such as cystic fibrosis, haemophilia A and B, chronicgranulomatous disease, X-linked severe combined immunodeficiency andfamilial hyperlipemia; (2) autosomal dominant syndromes; (3) many formsof cancer; (4) infectious diseases; (5) chronic inflammatory syndromes,and; (6) intractable pain. In the future, the therapy of diseasesassociated with multiple defects or pathogenetic mechanisms, such asdiabetes mellitus, may also become feasible.

Gene vaccination has been developed to cope with the poor protectionconferred by soluble proteins of a number of pathogens, includingviruses such as the human immunodeficiency virus (HIV). It was thoughtthat intracellular delivery of antigens could direct efficientprocessing into both major histocompatibility complexes (MHC) class Iand class II for improved activation of CD8+ and CD4+ T cells,respectively.

The host immune response towards viral vector proteins was soonrecognised as a limiting factor in gene therapy. Cells transduced withviral vectors elicit specific T cells, which lead to inflammation andcell lysis, and thereby aborting transgene expression. The results of arecent anti-HIV gene vaccination trial using recombinant adenovirusvectors expressing the HIV gag, pol or Nef gene were reported by Sekaly(2008), J. Exp. Med., 205, 7-12. Surprisingly, it was shown that thepresence of a pre-existing immune response towards viral vector proteinshad detrimental results on the outcome of vaccination. Thus, in bothsituations (i.e., either a pre-existing immune response or nopre-existing immune response) the immune response towards vector-relatedproteins appear to be ominous.

The immune response towards adenovirus provides one of the best examplesof this, as vectors derived from adenovirus are used in the setting ofboth gene therapy and gene vaccination. Adenovirus is highly immunogenicin man and mammals. Upon injection, adenoviruses elicit an acute innateimmune response, which results in inflammation and cytotoxicity, whichis often transient. This response, however, triggers an adaptiveresponse that leads to the activation of CD4+ and CD8+ T cells. This isobserved even with vectors from which most immunogenic proteins havebeen removed.

The adaptive immune response to adenovirus involves several components:specific antibodies, CD4+ and CD8+ T cells. Viral proteins are processedand presented by host antigen-presenting cells (APC) in the form ofpeptides bound to (MHC) of class I and II. Thus, such presentationresults in activation of specific T cells belonging to the CD8+ or CD4+subtype, respectively. The function of CD8+ T cells is to lyse cellsexpressing virus-derived MHC class I peptides. The function of CD4+ Tcells is multifaceted: helping B cells to mature and transform intoantibody-forming cells, helping CD8+ T cells to acquire full maturationand development of an inflammatory environment. As such, CD4+ specific Tcells play a central role in the elaboration of a virus-specific immuneresponse.

Adenoviruses are ubiquitous and more than 50 serotypes have beendescribed. Many subjects are therefore already immunised, which limitsthe use of vectors derived from such viruses.

Accordingly, in the setting of gene therapy as well as of genevaccination, it is highly desirable to find ways to prevent and/orsuppress immune responses to viral vector proteins.

SUMMARY OF THE INVENTION

The present invention relates in a first aspect to isolated immunogenicpeptides for use in preventing or suppressing in a recipient of a viralvector for gene therapy or gene vaccination, the immune responses tosaid viral vector. More particularly the invention relates to the use ofat least one isolated immunogenic peptide for the manufacture of amedicament for preventing or suppressing an immune response to a viralvector, in a recipient of said vector for gene therapy or genevaccination, the immunogenic peptide comprising (i) a T-cell epitopederived from a protein from said viral vector and (ii) a C-(X)2-[CST] or[CST]-(X)2-C motif.

In a further aspect, the invention relates to the use of at least oneisolated immunogenic peptide comprising (i) a T-cell epitope derivedfrom a viral vector protein and (ii) a [CST]-(X)2-[CST] motif, for themanufacture of a medicament for preventing, in a recipient of genetherapy or gene vaccination, activation of CD4+ effector T-cells by aviral vector protein.

In a further aspect, the invention also covers the use of at least oneisolated immunogenic peptide comprising (i) a T-cell epitope derivedfrom a viral vector protein and (ii) a [CST]-(X)2-[CST] motif, for themanufacture of a medicament for inducing, in a recipient of gene therapyor gene vaccination, CD4+ regulatory T cells which are cytotoxic tocells presenting a viral vector protein.

In a further aspect, the invention further relates to the use of atleast one isolated immunogenic peptide comprising (i) a T-cell epitopederived from a viral vector protein and (ii) [CST]-(X)2-[CST] motif, forthe manufacture of a medicament for preventing, in a recipient of genetherapy or gene vaccination, activation of CD8+ effector T-cells by aviral vector protein.

Generally, the invention provides immunogenic peptides comprising (i) aT-cell epitope derived from a viral vector protein and (ii) C-(X)2-[CST]or [CST]-(X)2-C motif for use in preventing or suppressing in arecipient of the viral vector (for gene therapy or gene vaccination) animmune response to the viral vector, preventing activation of CD4+and/or CD8+ effector T-cells of a recipient by a viral vector proteinand inducing in a recipient CD4+ regulatory T cells which are cytotoxicto cells presenting a viral vector protein (or epitope thereof).

In any of the above uses said viral vector protein may be derived fromadenovirus, adeno-associated virus, herpes virus or poxvirus or from aviral vector derived from any thereof. Alternatively, said viral vectorprotein is derived from retrovirus or lentivirus or from a viral vectorderived from any thereof.

In any of the above uses, said C-(X)2-[CST] or [CST]-(X)2-C motif insaid immunogenic peptide may be adjacent to said T-cell epitope, or beseparated from said T-cell epitope by a linker. In particularembodiments, the linker consists of at most 7 amino acids.

In a further embodiment to the immunogenic peptide in the above uses,the C-(X)2-[CST] or [CST]-(X)2-C motif does not naturally occur within aregion of 11 amino acids N- or C-terminally adjacent to the T-cellepitope in said viral vector protein. In particular the C-(X)2-[CST] or[CST]-(X)2-C motif is positioned N-terminally of the T-cell epitope.Further in particular embodiments, at least one X in said C-(X)2-[CST]or [CST]-(X)2-C motif is Gly, Ala, Ser or Thr; additionally oralternatively at least on X is His or Pro. In particular embodiments atleast one C in the C-(X)2-[CST] or [CST]-(X)2-C motif is methylated.

In particular embodiments of the immunogenic peptide envisaged for theabove uses, the immunogenic peptide further comprises an endosomaltargeting sequence. Any of the above immunogenic peptides may beproduced by chemical synthesis or by recombinant expression.

A further aspect of the invention relates to methods for obtaining apopulation of viral vector protein-specific regulatory T cells withcytotoxic properties, said methods comprising the steps of:

-   -   providing peripheral blood cells;    -   contacting these cells with an immunogenic peptide        comprising (i) a T-cell epitope derived from a viral vector        protein and (ii) a C-(X)2-[CST] or [CST]-(X)2-C motif; and    -   expanding these cells in the presence of IL-2.

A further method of the invention aims at obtaining a population ofviral vector protein-specific regulatory T cells with cytotoxicproperties, and such methods comprise the steps of:

-   -   providing an immunogenic peptide comprising (i) a T-cell epitope        derived from a viral vector protein and (ii) a C-(X)2-[CST] or        [CST]-(X)2-C motif;    -   administering the immunogenic peptide to a subject; and    -   obtaining a population of viral vector protein-specific        regulatory T cells from said subject.

Populations of viral vector protein-specific regulatory T cells withcytotoxic properties obtainable by the above methods are also part ofthe invention, as well as their use for the manufacture of a medicamentfor preventing or suppressing immune responses to viral vectors in arecipient of gene therapy or gene vaccination.

A further aspect of the invention relates to isolated immunogenicpeptides comprising a T-cell epitope from a viral vector protein and,adjacent to the T-cell epitope or separated from the T-cell epitope by alinker, a C-(X)2-[CST] or [CST]-(X)2-C motif.

The invention further encompasses isolated viral vectors characterisedin that they comprise at least one viral vector protein comprising aT-cell epitope and adjacent to the T-cell epitope or separated from theT-cell epitope by a linker, a C-(X)2-[CST] or [CST]-(X)2-C motif. Moreparticularly, the invention provides isolated viral vectorscharacterised in that at least one T-cell epitope present in at leastone of the viral vector proteins is modified by insertion in said viralvector protein, adjacent to said T-cell epitope or separated from saidT-cell epitope by a linker, of a C-(X)2-[CST] or [CST]-(X)2-C motif.

FIGURE LEGENDS

FIG. 1. Killing of splenic B cells with a T cell line specific for humanadenovirus 5 (HAdV-5). For detailed description, see Example 4.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “peptide” when used herein refers to a molecule comprising anamino acid sequence of between 2 and 200 amino acids, connected bypeptide bonds, but which can in a particular embodiment comprisenon-amino acid structures (like for example a linking organic compound).Peptides according to the invention can contain any of the conventional20 amino acids or modified versions thereof, or can containnon-naturally occurring amino acids incorporated by chemical peptidesynthesis or by chemical or enzymatic modification.

The term “epitope” when used herein refers to one or several portions(which may define a conformational epitope) of a protein which is/arespecifically recognised and bound by an antibody or a portion thereof(Fab′, Fab2′, etc.) or a receptor presented at the cell surface of a Bor T cell lymphocyte, and which is able, by said binding, to induce animmune response.

The term “antigen” when used herein refers to a structure of amacromolecule comprising one or more hapten(s) (eliciting an immuneresponse only when attached to a carrier) and/or comprising one or moreT cell epitopes. Typically, said macromolecule is a protein or peptide(with or without polysaccharides) or made of proteic composition andcomprises one or more epitopes; said macromolecule can hereinalternatively be referred to as “antigenic protein” or “antigenicpeptide”.

“Gene therapy” can be defined as the insertion, ex vivo or in vivo, of agene or genes into individual cells or groups of cells (such as tissuesor organs), whereby expression of the gene in the cells or groups ofcells ensures a therapeutic effect. In many cases gene therapy iscarried out to provide a missing gene or allele or to replace a mutantgene or a mutant allele with a functional copy. The “therapeutic gene”is delivered via a carrier called a vector. The most common vector is aviral vector. Upon infection of targeted cells with the viral vectorcarrying the therapeutic gene, the viral vector unloads its geneticmaterial including the therapeutic gene into the target cells, followedby the generation of the functional protein(s) encoded by thetherapeutic gene. Cells targeted by gene therapy can be either somaticcells or germ cells or cell lines. In addition, gene therapy refers tothe use of vectors to deliver, either ex vivo or in vivo, a gene thatrequires overexpression or ectopic expression in a cell or group ofcells. The vector can facilitate integration of the new gene in thenucleus or can lead to episomal expression of that gene.

“Gene vaccination” can be defined as the administration of a functionalgene (i.e., capable of expressing the protein encoded by the gene) to asubject for the purpose of vaccinating said subject. Thus, genevaccination (or DNA vaccination) is a variant of the more classicalvaccination with peptides, proteins, attenuated or killed germs, etc.Gene vaccination can be performed with naked DNA or, of particularinterest in the context of the present invention, with viral vectors.

The term “viral vector protein” when used herein refers to any proteinor peptide derived from a viral vector. Typically such proteins areantigenic and comprise one or more epitopes such as T-cell epitopes.

The term “T cell epitope” or “T-cell epitope” in the context of thepresent invention refers to a dominant, sub-dominant or minor T cellepitope, i.e., a part of an antigenic protein that is specificallyrecognised and bound by a receptor at the cell surface of a Tlymphocyte. Whether an epitope is dominant, sub-dominant or minordepends on the immune reaction elicited against the epitope. Dominancedepends on the frequency at which such epitopes are recognised by Tcells and able to activate them, among all the possible T cell epitopesof a protein. In particular, a T cell epitope is an epitope bound by MHCclass I or MHC class II molecules.

The term “MHC” refers to “major histocompatibility antigen”. In humans,the MHC genes are known as HLA (“human leukocyte antigen”) genes.Although there is no consistently followed convention, some literatureuses HLA to refer to HLA protein molecules, and MHC to refer to thegenes encoding the HLA proteins. As such the terms “MHC” and “HLA” areequivalents when used herein. The HLA system in man has its equivalentin the mouse, i.e., the H2 system. The most intensely-studied HLA genesare the nine so-called classical MHC genes: HLA-A, HLA-B, HLA-C,HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1. Inhumans, the MHC is divided into three regions: Class I, II, and III. TheA, B, and C genes belong to MHC class I, whereas the six D genes belongto class II. MHC class I molecules are made of a single polymorphicchain containing 3 domains (alpha 1, 2 and 3), which associates withbeta 2 microglobulin at cell surface. Class II molecules are made of 2polymorphic chains, each containing 2 chains (alpha 1 and 2, and beta 1and 2).

Class I MHC molecules are expressed on virtually all nucleated cells.Peptide fragments presented in the context of class I MHC molecules arerecognised by CD8+ T lymphocytes (cytotoxic T lymphocytes or CTLs). CD8+T lymphocytes frequently mature into cytotoxic effectors which can lysecells bearing the stimulating antigen. Class II MHC molecules areexpressed primarily on activated lymphocytes and antigen-presentingcells. CD4+ T lymphocytes (helper T lymphocytes or HTLs) are activatedwith recognition of a unique peptide fragment presented by a class IIMHC molecule, usually found on an antigen presenting cell like amacrophage or dendritic cell. CD4+ T lymphocytes proliferate and secretecytokines that either support an antibody-mediated response through theproduction of IL-4 and IL-10 or support a cell-mediated response throughthe production of IL-2 and IFN-gamma.

Functional HLAs are characterised by a deep binding groove to whichendogenous as well as foreign, potentially antigenic peptides bind. Thegroove is further characterised by a well-defined shape andphysico-chemical properties. HLA class I binding sites are closed, inthat the peptide termini are pinned down into the ends of the groove.They are also involved in a network of hydrogen bonds with conserved HLAresidues. In view of these restraints, the length of bound peptides islimited to 8-10 residues. However, it has been demonstrated thatpeptides of up to 12 amino acid residues are also capable of binding HLAclass I. Superposition of the structures of different HLA complexesconfirmed a general mode of binding wherein peptides adopt a relativelylinear, extended conformation.

In contrast to HLA class I binding sites, class II sites are open atboth ends. This allows peptides to extend from the actual region ofbinding, thereby “hanging out” at both ends. Class II HLAs can thereforebind peptide ligands of variable length, ranging from 9 to more than 25amino acid residues. Similar to HLA class I, the affinity of a class IIligand is determined by a “constant” and a “variable” component. Theconstant part again results from a network of hydrogen bonds formedbetween conserved residues in the HLA class II groove and the main-chainof a bound peptide. However, this hydrogen bond pattern is not confinedto the N- and C-terminal residues of the peptide but distributed overthe whole chain. The latter is important because it restricts theconformation of complexed peptides to a strictly linear mode of binding.This is common for all class II allotypes. The second componentdetermining the binding affinity of a peptide is variable due to certainpositions of polymorphism within class II binding sites. Differentallotypes form different complementary pockets within the groove,thereby accounting for subtype-dependent selection of peptides, orspecificity. Importantly, the constraints on the amino acid residuesheld within class II pockets are in general “softer” than for class I.There is much more cross reactivity of peptides among different HLAclass II allotypes. The sequence of the +/−9 amino acids of an MHC classII T cell epitope that fit in the groove of the MHC II molecule areusually numbered P1 to P9. Additional amino acids N-terminal of theepitope are numbered P−1, P−2 and so on, amino acids C-terminal of theepitope are numbered P+1, P+2 and so on.

The term “organic compound having a reducing activity” when used hereinrefers to compounds, more in particular amino acid sequences, capable ofreducing disulfide bonds in proteins. An alternatively used term forthese amino acid sequences is “redox motif”.

The term “therapeutically effective amount” refers to an amount of thepeptide of the invention or derivative thereof, which produces thedesired therapeutic or preventive effect in a patient. For example, inreference to a disease or disorder, it is the amount which reduces tosome extent one or more symptoms of the disease or disorder, and moreparticularly returns to normal, either partially or completely, thephysiological or biochemical parameters associated with or causative ofthe disease or disorder. According to one particular embodiment of thepresent invention, the therapeutically effective amount is the amount ofthe peptide of the invention or derivative thereof, which will lead toan improvement or restoration of the normal physiological situation. Forinstance, when used to therapeutically treat a mammal affected by animmune disorder, it is a daily amount peptide/kg body weight of the saidmammal. Alternatively, where the administration is through gene-therapy,the amount of naked DNA or viral vectors is adjusted to ensure the localproduction of the relevant dosage of the peptide of the invention,derivative or homologue thereof.

The term “natural” when used herein referring to a sequence relates tothe fact that the sequence is identical to a naturally occurringsequence or is identical to part of such naturally occurring sequence.In contrast therewith the term “artificial” refers to a sequence whichas such does not occur in nature. Unless otherwise specified, the termsnatural and artificial thus exclusively relate to a particular aminoacid (or nucleotide) sequence (e.g. the sequence of the immunogenicpeptide, a sequence comprised within the immunogenic peptide en epitopesequence) and do not refer to the nature of the immunogenic peptide assuch. Optionally, an artificial sequence is obtained from a naturalsequence by limited modifications such as changing one or more aminoacids within the naturally occurring sequence or by adding amino acidsN- or C-terminally of a naturally occurring sequence. Amino acids arereferred to herein with their full name, their three-letter abbreviationor their one letter abbreviation.

Motifs of amino acid sequences are written herein according to theformat of Prosite (Hulo et al. (2006) Nucleic Acids Res. 34 (Databaseissue D227-D230). The symbol X is used for a position where any aminoacid is accepted. Alternatives are indicated by listing the acceptableamino acids for a given position, between square brackets (‘[ ]’). Forexample: [CST] stands for an amino acid selected from Cys, Ser or Thr.Amino acids which are excluded as alternatives are indicated by listingthem between curly brackets (‘{ }’). For example: {AM} stands for anyamino acid except Ala and Met. The different elements in a motif areseparated from each other by a hyphen -. Repetition of an identicalelement within a motif can be indicated by placing behind that element anumerical value or a numerical range between parentheses. For example:X(2) corresponds to X-X, X(2, 4) corresponds to X-X or X-X-X or X-X-X-X,A(3) corresponds to A-A-A.

The term “homologue” when used herein with reference to the epitopesused in the context of the invention, refer to molecules having at least50%, at least 70%, at least 80%, at least 90%, at least 95% or at least98% amino acid sequence identity with the naturally occurring epitopesequence, thereby maintaining the ability of the epitope to bind anantibody or cell surface receptor of a B and/or T cell. Particularembodiments of homologues of an epitope correspond to the naturalepitope sequence modified in at most three, more particularly in at mosttwo, most particularly in one amino acid.

The term “derivative” when used herein with reference to the peptides ofthe invention refers to molecules which contain at least the peptideactive portion (i.e. capable of eliciting cytolytic CD4+ T cellactivity) and, in addition thereto comprises a complementary portionwhich can have different purposes such as stabilising the peptides oraltering the pharmacokinetic or pharmacodynamic properties of thepeptide.

The term “sequence identity” of two sequences when used herein relatesto the number of positions with identical nucleotides or amino acidsdivided by the number of nucleotides or amino acids in the shorter ofthe sequences, when the two sequences are aligned. In particularembodiments, said sequence identity is from 70% to 80%, from 81% to 85%,from 86% to 90%, from 91% to 95%, from 96% to 100%, or 100%.

The terms “peptide-encoding polynucleotide (or nucleic acid)” and“polynucleotide (or nucleic acid) encoding peptide” when used hereinrefer to a nucleotide sequence, which, when expressed in an appropriateenvironment, results in the generation of the relevant peptide sequenceor a derivative or homologue thereof. Such polynucleotides or nucleicacids include the normal sequences encoding the peptide, as well asderivatives and fragments of these nucleic acids capable of expressing apeptide with the required activity. According to one embodiment, thenucleic acid encoding the peptides according to the invention orfragment thereof is a sequence encoding the peptide or fragment thereoforiginating from a mammal or corresponding to a mammalian, mostparticularly a human peptide fragment.

The present invention provides ways to prevent and/or suppress immuneresponses to proteins derived from viral vectors as used in gene therapyand gene vaccination. In particular, the invention provides ways toprevent the development of and/or suppress a CD4+ effector T cells(alternatively referred to as bystander T cells) response. Instead CD4+regulatory T cells are induced which are capable of specificallyinducing apoptosis of APCs presenting T cell epitopes processed fromviral vector proteins, thereby preventing the formation of specificantibodies, preventing the maturation of CD8+ T cells and reducing theinflammatory consequences of the proliferation of CD4+ T cells. Aconsequence of the prevention of full maturation of CD8+ T cellsincludes the prevention of cytolysis of virally-transduced cells throughMHC class I presentation of viral vector-derived peptides. The compoundsused to achieve the above are immunogenic peptides encompassing thesequence of a T cell epitope derived from the processing of viral vectorproteins attached to a redox motif such as C-(X)2-C. The T cell epitopemodified in this way alters the activation pattern and function of CD4+T cells, either de novo from naïve T cells in a prevention setting, orby modifying the properties of memory T cells, both resulting in potentcapacity to induce apoptosis of APC. Thereby the antibody and cellularresponses towards viral vector proteins are prevented and/or suppressed.More specifically, the elimination of an APC (dendritic cells, B cellsor macrophages, in the setting of primary and secondary immuneresponses, respectively) presenting MHC class II bound peptidesprocessed from viral vector proteins results in tolerance induction toviral vector proteins. Hence, a major obstacle for efficient genetherapy or gene vaccination is cleared by using the above-describedcompounds.

In a first aspect the invention relates to isolated immunogenic peptidesfor use in preventing or suppressing, in a recipient of a viral vectore.g. for gene therapy or gene vaccination, the immune responses to saidviral vector. More particularly the invention envisages the use of atleast one isolated immunogenic peptide comprising (i) a T-cell epitopederived from a viral vector protein and (ii) a C-(X)2-[CST] or[CST]-(X)2-C motif, for the manufacture of a medicament for preventingor suppressing, in a recipient of gene therapy or gene vaccination, theimmune responses to said viral vector. Hence, said immunogenic peptideor the medicament comprising it can be used for prior or prophylactictreatment or immunisation of a recipient of gene therapy or genevaccination in order to suppress, avoid, reduce partially or totally, oreliminate (partially or totally) immune response(s) induced by thesubsequently applied gene therapy or gene vaccination. Likewise,immunogenic peptides according to the invention or the medicamentscomprising them can be used for therapeutic treatment or immunisation ofa recipient of gene therapy or gene vaccination in order to suppress,reduce partially or totally, or eliminate (partially or totally) ongoingimmune response(s) to a viral vector induced by said gene therapy orgene vaccination.

In a further aspect, the invention relates to the use of at least oneisolated immunogenic peptide comprising (i) a T-cell epitope derivedfrom a viral vector protein and (ii) a C-(X)2-[CST] or [CST]-(X)2-Cmotif, for the manufacture of a medicament for preventing, in arecipient of gene therapy or gene vaccination, activation of CD4+effector T-cells by a viral vector protein.

In a further aspect, the invention also covers the use of at least oneisolated immunogenic peptide comprising (i) a T-cell epitope derivedfrom a viral vector protein and (ii) a [CST]-(X)2-[CST]C-(X)2-[CST] or[CST]-(X)2-C motif, for the manufacture of a medicament for inducing, ina recipient of gene therapy or gene vaccination, CD4+ regulatory T cellswhich are cytotoxic to cells presenting a viral vector protein.

In a further aspect, the invention further relates to the use of atleast one isolated immunogenic peptide comprising (i) a T-cell epitopederived from a viral vector protein and (ii) C-(X)2-[CST] or[CST]-(X)2-C motif, for the manufacture of a medicament for preventing,in a recipient of gene therapy or gene vaccination, (full) activation ormaturation of CD8+ effector T-cells by a viral vector protein.

In the above aspects of the invention, immunogenic peptides according tothe invention or the medicaments comprising them can be used for prioror prophylactic treatment or immunisation of a recipient of gene therapyor gene vaccination in order to suppress, avoid, reduce partially ortotally, or eliminate (partially or totally) a normally expectedactivation in the recipient of CD4+ effector T-cells and/or CD8+ T-cellstowards the viral vector following or subsequent to the actual genetherapy or gene vaccination. Likewise, immunogenic peptides according tothe invention or medicament comprising them can be used for therapeutictreatment or immunisation of a recipient of gene therapy or genevaccination in order to suppress, reduce partially or totally, oreliminate (partially or totally) activation in the recipient of CD4+effector T-cells and/or CD8+ T-cells towards the viral vector concurrentwith or after the actual gene therapy or gene vaccination.Alternatively, or concurrently with any of the above, immunogenicpeptides according to the invention or the medicaments comprising themcan be used for prior or prophylactic treatment or immunisation of arecipient of gene therapy or gene vaccination in order to induce anormally unexpected activation in the recipient of viral vectorprotein-specific CD4+ regulatory T-cells capable of killing cellspresenting viral vector antigen(s) following or subsequent to the actualgene therapy or gene vaccination. Likewise, immunogenic peptidesaccording to the invention or the medicaments comprising them can beused for therapeutic treatment or immunisation of a recipient of genetherapy or gene vaccination in order to induce activation in therecipient of viral vector antigen-specific CD4+ regulatory T-cellscapable of killing cells presenting viral vector antigen(s). Saidinduction may happen concurrent with or after the actual gene therapy orgene vaccination.

In any of the uses described hereinabove, the recipient is a mammal, inparticular a (non-human) primate or a human.

In any of the above uses said viral vector protein may be a viralprotein derived from adenovirus, adeno-associated virus, herpes virus orpoxvirus or from a viral vector derived from any thereof. Alternatively,the viral vector protein is derived from retrovirus (such asgamma-retrovirus) or lentivirus or from a viral vector derived from anythereof. In particular embodiments the viral vector protein is a proteinpresent in the viral vector. In particular embodiments the viral proteinis a viral protein (encoded by viral DNA).

The cytotoxic regulatory T cells elicited by the immunogenic peptides ofthe present invention can suppress immune responses to even complexviral vector antigens. A minimum requirement for such cells to beactivated is to recognise a cognate peptide presented by MHC class IIdeterminants, leading to apoptosis of the APC, thereby suppressing theresponses of T cells (both CD4+ and CD8+ T cells) to all T cell epitopespresented by the APC. An additional mechanism by which cytotoxicregulator T cells can suppress the overall immune response towardscomplex antigens is by suppressing the activation of bystander T cells.

There are situations in which more than one viral vector antigencontributes to the immune response against the viral vector. Under suchcircumstances, the same APC may not present all relevant viral vectorantigens, as some of such antigens may be taken up by potentiallydifferent APCs. It is therefore anticipated that combination of two ormore immunogenic peptides may be used for the prevention and suppressionof immune responses to a viral vector.

In any of the uses and methods described hereinabove, the immunogenicpeptides can be replaced by CD4+ regulatory T-cells primed with theimmunogenic peptide, or can be replaced by a nucleotide sequenceencoding the immunogenic peptide (e.g. in the form of naked DNA or aviral vector to be administered to an individual instead of theimmunogenic peptide). In addition, a combination of multiple immunogenicpeptides, i.e. more than 1 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more),can be used in any of the above. These aspects of the invention, as wellas the further modification of the immunogenic peptide are described indetail hereafter.

The present invention is based upon the finding that an immunogenicpeptide, comprising a T cell epitope derived from a viral vector antigenand a peptide sequence having reducing activity is capable of generatinga population of CD4+ regulatory T cells, which have a cytotoxic effecton antigen presenting cells. It is additionally based upon the findingthat such immunogenic peptide is capable of preventing activation ofviral vector antigen-specific CD8+ T cells and/or CD4+ effector T cells.

Accordingly, the invention relates to immunogenic peptides, whichcomprise at least one T-cell epitope of a viral vector antigen with apotential to trigger an immune reaction, coupled to an organic compoundhaving a reducing activity, such as a thioreductase sequence motif. TheT cell epitope and the organic compound are optionally separated by alinker sequence. In further optional embodiments the immunogenic peptideadditionally comprises an endosome targeting sequence (e.g. lateendosomal targeting sequence) and/or additional “flanking” sequences.

The immunogenic peptides of the invention can be schematicallyrepresented as A-L-B or B-L-A, wherein A represents a T-cell epitope ofan antigen (of a viral vector protein) with a potential to trigger animmune reaction, L represents a linker and B represents an organiccompound having a reducing activity.

The reducing activity of an organic compound can be assayed for itsability to reduce a sulfhydryl group such as in the insulin solubilityassay known in the art, wherein the solubility of insulin is alteredupon reduction, or with a fluorescence-labelled insulin. The reducingorganic compound may be coupled at the amino-terminus side of the T-cellepitope or at the carboxy-terminus of the T-cell epitope.

Generally the organic compound with reducing activity is a peptidesequence. Peptide fragments with reducing activity are encountered inthioreductases which are small disulfide reducing enzymes includingglutaredoxins, nucleoredoxins, thioredoxins and other thiol/disulfideoxidoreductases They exert reducing activity for disulfide bonds onproteins (such as enzymes) through redox active cysteines withinconserved active domain consensus sequences: C-X(2)-C, C-X(2)-S,C-X(2)-T, S-X(2)-C, T-X(2)-C (Fomenko et al. (2003) Biochemistry 42,11214-11225), in which X stands for any amino acid. Such domains arealso found in larger proteins such as protein disulfide isomerase (PDI)and phosphoinositide-specific phospholipase C.

Accordingly, in particular embodiments, immunogenic peptides accordingto the present invention comprise as redox motif the thioreductasesequence motif [CST]-X(2)-[CST], in a further embodiment thereto, the[CST]-X(2)-[CST] motif is positioned N-terminally of the T-cell epitope.More specifically, in the redox motif at least one of the [CST]positions is occupied by a Cys; thus the motif is either [C]-X(2)-[CST]or [CST]-X(2)-[C]. In the present application such a tetrapeptide willbe referred to as “the motif”. In particular embodiments peptides of theinvention contain the sequence motif [C]-X(2)-[CS] or [CS]-X(2)-[C]. Inmore particular embodiments peptides contain the sequence motifC-X(2)-S, S-X(2)-C or C-X(2)-C.

As explained in detail further on, the immunogenic peptides of thepresent invention can be made by chemical synthesis, which allows theincorporation of non-natural amino acids. Accordingly, in the motif ofreducing compounds according to particular embodiments of the presentinvention, C represents either cysteine or another amino acids with athiol group such as mercaptovaline, homocysteine or other natural ornon-natural amino acids with a thiol function. In order to have reducingactivity, the cysteines present in the motif should not occur as part ofa cystine disulfide bridge. Nevertheless, the motif may comprisemodified cysteines such as methylated cysteine, which is converted intocysteine with free thiol groups in vivo.

Either of the amino acids X in the C-(X)2-[CST] or [CST]-(X)2-C motif ofparticular embodiments of the immunogenic peptides of the invention canbe any natural amino acid, including S, C, or T or can be a non-naturalamino acid. In particular embodiments X is an amino acid with a smallside chain such as Gly, Ala, Ser or Thr. In further particularembodiments, X is not an amino acid with a bulky side chain such as Tyr.In further particular embodiments at least one X in the [CST]-X(2)-[CST]motif is His or Pro.

In the immunogenic peptides of the present invention comprising the(redox) motif described above, the motif is located such that, when theepitope fits into the MHC groove, the motif remains outside of the MHCbinding groove. The motif is placed either immediately adjacent to theepitope sequence within the peptide, or is separated from the T cellepitope by a linker. More particularly, the linker comprises an aminoacid sequence of 7 amino acids or less. Most particularly, the linkercomprises 1, 2, 3, or 4 amino acids. Alternatively, a linker maycomprise 6, 8 or 10 amino acids. Typical amino acids used in linkers areserine and threonine. Example of peptides with linkers in accordancewith the present invention are CXXC-G-epitope (SEQ ID NO:1),CXXC-GG-epitope (SEQ ID NO:2), CXXC-SSS-epitope (SEQ ID NO:3),CXXC-SGSG-epitope (SEQ ID NO:4) and the like.

In those particular embodiments of the peptides of the invention wherethe motif sequence is adjacent to the epitope sequence this is indicatedas position P−4 to P−1 or P+1 to P+4 compared to the epitope sequence.Apart from a peptide linker other organic compounds can be used aslinker to link the parts of the immunogenic peptide to each other.

The immunogenic peptides of the present invention can further compriseadditional short amino acid sequences N or C-terminally of the(artificial) sequence comprising the T cell epitope and the reducingcompound (motif). Such an amino acid sequence is generally referred toherein as a ‘flanking sequence’. A flanking sequence can be positionedN- and/or C-terminally of the redox motif and/or of the T-cell epitopein the immunogenic peptide. When the immunogenic peptide comprises anendosomal targeting sequence, a flanking sequence can be present betweenthe epitope and an endosomal targeting sequence and/or between thereducing compound (e.g. motif) and an endosomal targeting sequence. Moreparticularly a flanking sequence is a sequence of up to 10 amino acids,or of in between 1 and 7 amino acids, such as a sequence of 2 aminoacids.

In particular embodiments of the invention, the redox motif in theimmunogenic peptide is located N-terminally from the epitope.

In further particular embodiments, where the redox motif present in theimmunogenic peptide contains one cysteine, this cysteine is present inthe motif in the position most remote from the epitope, thus the motifoccurs as C-X(2)-[ST] or C-X(2)-S N-terminally of the epitope or occursas [ST]-X(2)-C or S-X(2)-C carboxy-terminally of the epitope.

In certain embodiments of the present invention, immunogenic peptidesare provided comprising one epitope sequence and a motif sequence. Infurther particular embodiments, the motif occurs several times (1, 2, 3,4 or even more times) in the peptide, for example as repeats of themotif which can be spaced from each other by one or more amino acids(e.g. CXXC X CXXC X CXXC; SEQ ID NO:5), as repeats which are adjacent toeach other (CXXC CXXC CXXC; SEQ ID NO:6) or as repeats which overlapwith each other CXXCXXCXXC (SEQ ID NO:7) or CXCCXCCXCC (SEQ ID NO:8)).Alternatively, one or more motifs are provided at both the N and the Cterminus of the T cell epitope sequence. Other variations envisaged forthe immunogenic peptides of the present invention include peptidescontaining repeats of a T cell epitope sequence or multiple differentT-cell epitopes wherein each epitope is preceded and/or followed by themotif (e.g. repeats of “motif-epitope” or repeats of“motif-epitope-motif”). Herein the motifs can all have the same sequencebut this is not obligatory. It is noted that repetitive sequences ofpeptides which comprise an epitope which in itself comprises the motifwill also result in a sequence comprising both the ‘epitope’ and a‘motif’. In such peptides, the motif within one epitope sequencefunctions as a motif outside a second epitope sequence. In particularembodiments however, the immunogenic peptides of the present inventioncomprise only one T cell epitope.

As described above the immunogenic peptides according to the inventioncomprise, in addition to a reducing compound/motif, a T cell epitopederived from a viral vector antigen. A T cell epitope in a proteinsequence can be identified by functional assays and/or one or more insilico prediction assays. The amino acids in a T cell epitope sequenceare numbered according to their position in the binding groove of theMHC proteins. In particular embodiments, the T-cell epitope presentwithin the peptides of the invention consists of between 8 and 25 aminoacids, yet more particularly of between 8 and 16 amino acids, yet mostparticularly consists of 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids.In a more particular embodiment, the T cell epitope consists of asequence of 9 amino acids. In a further particular embodiment, theT-cell epitope is an epitope, which is presented to T cells by MHC-classII molecules. In particular embodiments of the present invention, the Tcell epitope sequence is an epitope sequence which fits into the cleftof an MHC II protein, more particularly a nonapeptide fitting into theMHC II cleft. The T cell epitope of the immunogenic peptides of theinvention can correspond either to a natural epitope sequence of aprotein or can be a modified version thereof, provided the modified Tcell epitope retains its ability to bind within the MHC cleft, similarto the natural T cell epitope sequence. The modified T cell epitope canhave the same binding affinity for the MHC protein as the naturalepitope, but can also have a lowered affinity. In particular embodimentsthe binding affinity of the modified peptide is no less than 10-foldless than the original peptide, more particularly no less than 5 timesless. It is a finding of the present invention that the peptides of thepresent invention have a stabilising effect on protein complexes.Accordingly, the stabilising effect of the peptide-MHC complexcompensates for the lowered affinity of the modified epitope for the MHCmolecule.

In particular embodiments, the immunogenic peptides of the inventionfurther comprise an amino acid sequence (or another organic compound)facilitating uptake of the peptide into (late) endosomes for processingand presentation within MHC class II determinants. The late endosometargeting is mediated by signals present in the cytoplasmic tail ofproteins and correspond to well-identified peptide motifs such as thedileucine-based [DE]XXXL[LI] (SEQ ID NO:9) or DXXLL (SEQ ID NO:10)-motif(e.g. DXXXLL; SEQ ID NO:11), the tyrosine-based YXXØ motif or theso-called acidic cluster motif. The symbol Ø represents amino acidresidues with a bulky hydrophobic side chains such as Phe, Tyr and Trp.The late endosome targeting sequences allow for processing and efficientpresentation of the antigen-derived T cell epitope by MHC-class IImolecules. Such endosomal targeting sequences are contained, forexample, within the gp75 protein (Vijayasaradhi et al. (1995) J CellBiol 130, 807-820), the human CD3 gamma protein, the HLA-BM β (Copier etal. (1996) J. Immunol. 157, 1017-1027), the cytoplasmic tail of theDEC205 receptor (Mahnke et al. (2000) J Cell Biol 151, 673-683). Otherexamples of peptides which function as sorting signals to the endosomeare disclosed in the review of Bonifacio and Traub (2003) Annu. Rev.Biochem. 72, 395-447. Alternatively, the sequence can be that of asubdominant or minor T cell epitope from a protein, which facilitatesuptake in late endosome without overcoming the T cell response towardsthe viral vector protein-derived T cell epitope.

The immunogenic peptides of the invention can be generated by coupling areducing compound, more particularly a reducing motif as describedherein, N-terminally or C-terminally to a T-cell epitope of the viralvector antigenic protein (either directly adjacent thereto or separatedby a linker). Moreover the T cell epitope sequence of the immunogenicpeptide and/or the redox motif can be modified and/or one or moreflanking sequences and/or a targeting sequence can be introduced (ormodified), compared to the naturally occurring T-cell epitope sequence.Accordingly, the resulting sequence of the immunogenic peptide will inmost cases differ from the sequence of the viral vector antigenicprotein of interest. In this case, the immunogenic peptides of theinvention are peptides with an ‘artificial’, non-naturally occurringsequence.

The immunogenic peptides of the invention can vary substantially inlength, e.g. from about 12-13 amino acids (a T-cell epitope of 8-9 aminoacids and the 4-amino acid redox motif) to up to 50 or more amino acids.For example, an immunogenic peptide according to the invention maycomprise an endosomal targeting sequence of 40 amino acids, a flankingsequence of about 2 amino acids, a motif as described herein of 4 aminoacids, a linker of 4 amino acids and a T cell epitope peptide of 9 aminoacids. In particular embodiments, the immunogenic peptides of theinvention consist of between 12 amino acids and 20 up to 25, 30, 50, 75,100 or 200 amino acids. In a more particular embodiment, the peptidesconsist of between 10 and 20 amino acids. More particularly, where thereducing compound is a redox motif as described herein, the length ofthe immunogenic peptide comprising the epitope and motif optionallyconnected by a linker is 19 amino acids or less, e.g., 12, 13, 14, 15,16, 17, 18 or 19 amino acids.

As detailed above, the immunogenic peptides of the invention comprise areducing motif as described herein linked to a T cell epitope sequence.According to particular embodiments the T-cell epitopes are derived fromviral vector proteins which do not comprise within their native naturalsequence an amino acid sequence with redox properties within a sequenceof 11 amino acids N- or C- terminally adjacent to the T-cell epitope ofinterest. Most particularly, the invention encompasses generatingimmunogenic peptides from viral vector antigenic proteins which do notcomprise a sequence selected from C-X(2)-S, S-X(2)-C, C-X(2)-C,S-X(2)-S, C-X(2)-T, T-X(2)-C within a sequence of 11 amino acids N- orC-terminally adjacent to the epitope sequence. In further particularembodiments, the present invention provides immunogenic peptides ofviral vector antigenic proteins which do not comprise theabove-described amino acid sequences with redox properties within theirsequence.

In further particular embodiments, the immunogenic peptides of theinvention are peptides comprising T cell epitopes, which T cell epitopesdo not comprise an amino acid sequence with redox properties withintheir natural sequence. However, in alternative embodiments, a T cellepitope binding to the MHC cleft may comprise a redox motif such asdescribed herein within its epitope sequence; the immunogenic peptidesaccording to the invention comprising such T-cell epitope must furthercomprise another redox motif coupled (adjacent of separated by a linker)N- or C-terminally to the epitope such that the attached motif canensure the reducing activity (contrary to the motif present in theepitope, which is buried within the cleft).

Another aspect of the present invention relates to methods forgenerating immunogenic peptides of the present invention describedherein. Such methods include the identification of T-cell epitopes in aviral vector antigenic protein of interest; ways for in vitro and insilico identification T-cell epitopes are amply known in the art andsome aspects are elaborated upon hereafter.

In particular embodiments, methods according to the invention includethe generation of immunogenic peptides of the invention including theidentified T-cell epitope and a redox motif (with or without linker(s),flanking sequence(s) or endosomal targeting sequence). The generatedimmunogenic peptides can be assessed for the capability to induce viralvector protein-specific CD4+ regulatory T cells which are cytotoxic forcells presenting (parts of) the viral vector antigenic protein ofinterest.

Immunogenic peptides according to the invention are generated startingfrom T cell epitope(s) of the viral vector protein(s) of interest. Inparticular, the T-cell epitope used may be a dominant T-cell epitope.The identification and selection of a T-cell epitope from viral vectorproteins, for use in the context of the present invention is known to aperson skilled in the art. For instance, peptide sequences isolated froma viral vector protein are tested by, for example, T cell biologytechniques, to determine whether the peptide sequences elicit a T cellresponse. Those peptide sequences found to elicit a T cell response aredefined as having T cell stimulating activity. Human T cell stimulatingactivity can further be tested by culturing T cells obtained from anindividual sensitised to a viral vector antigen with a peptide/epitopederived from the viral vector antigen and determining whetherproliferation of T cells occurs in response to the peptide/epitope asmeasured, e.g., by cellular uptake of tritiated thymidine. Stimulationindices for responses by T cells to peptides/epitopes can be calculatedas the maximum CPM in response to a peptide/epitope divided by thecontrol CPM. A T cell stimulation index (S.I.) equal to or greater thantwo times the background level is considered “positive.” Positiveresults are used to calculate the mean stimulation index for eachpeptide/epitope for the group of peptides/epitopes tested. Non-natural(or modified) T-cell epitopes can further optionally be tested for theirbinding affinity to MHC class II molecules. The binding of non-natural(or modified) T-cell epitopes to MHC class II molecules can be performedin different ways. For instance, soluble HLA class II molecules areobtained by lysis of cells homozygous for a given class II molecule. Thelatter is purified by affinity chromatography. Soluble class IImolecules are incubated with a biotin-labelled reference peptideproduced according to its strong binding affinity for that class IImolecule. Peptides to be assessed for class II binding are thenincubated at different concentrations and their capacity to displace thereference peptide from its class II binding is calculated by addition ofneutravidin. Methods can be found in for instance Texier et al., (2000)J. Immunology 164, 3177-3184). The immunogenic peptides of the inventionhave a mean T cell stimulation index of greater than or equal to 2.0. Animmunogenic peptide having a T cell stimulation index of greater than orequal to 2.0 is considered useful as a prophylactic or therapeuticagent. More particularly, immunogenic peptides according to theinvention have a mean T cell stimulation index of at least 2.5, at least3.5, at least 4.0, or even at least 5.0. In addition, such peptidestypically have a positivity index (P.I.) of at least about 100, at least150, at least about 200 or at least about 250. The positivity index fora peptide is determined by multiplying the mean T cell stimulation indexby the percent of individuals, in a population of individuals sensitiveto a viral vector antigen (e. g., at least 9 individuals, at least 16individuals or at least 29 or 30, or even more), who have T cells thatrespond to the peptide (thus corresponding to the SI multiplied by thepromiscuous nature of the peptide/epitope). Thus, the positivity indexrepresents both the strength of a T cell response to a peptide (S.I.)and the frequency of a T cell response to a peptide in a population ofindividuals sensitive to a viral vector antigen. In order to determineoptimal T cell epitopes by, for example, fine mapping techniques, apeptide having T cell stimulating activity and thus comprising at leastone T cell epitope as determined by T cell biology techniques ismodified by addition or deletion of amino acid residues at either the N-or C-terminus of the peptide and tested to determine a change in T cellreactivity to the modified peptide. If two or more peptides which sharean area of overlap in the native protein sequence are found to havehuman T cell stimulating activity, as determined by T cell biologytechniques, additional peptides can be produced comprising all or aportion of such peptides and these additional peptides can be tested bya similar procedure. Following this technique, peptides are selected andproduced recombinantly or synthetically. T cell epitopes or peptides areselected based on various factors, including the strength of the T cellresponse to the peptide/epitope (e.g., stimulation index) and thefrequency of the T cell response to the peptide in a population ofindividuals.

Candidate antigens can be screened by one or more in vitro algorithms toidentify a T cell epitope sequence within an antigenic protein. Suitablealgorithms are described for example in Zhang et al. (2005) NucleicAcids Res 33, W180-W183 (PREDBALB); Salomon & Flower (2006) BMCBioinformatics 7, 501 (MHCBN); Schuler et al. (2007) Methods Mol Biol.409, 75-93 (SYFPEITHI); Dönnes & Kohlbacher (2006) Nucleic Acids Res.34, W194-W197 (SVMHC); Kolaskar & Tongaonkar (1990) FEBS Lett. 276,172-174 and Guan et al. (2003) Appl Bioinformatics 2, 63-66 (MHCPred).More particularly, such algorithms allow the prediction within anantigenic protein of one or more nonapeptide sequences which will fitinto the groove of an MHC II molecule.

The immunogenic peptides of the invention can be produced by recombinantexpression in, e.g., bacterial cells (e.g. Escherichia coli), yeastcells (e.g., Pichia species, Hansenula species, Saccharomyces orSchizosaccharomyces species), insect cells (e.g. from Spodopterafrugiperda or Trichoplusia ni), plant cells or mammalian cells (e.g.,CHO, COS cells). The construction of the therefore required suitableexpression vectors (including further information such as promoter andtermination sequences) involves standard recombinant DNA techniques.Recombinantly produced immunogenic peptides of the invention can bederived from a larger precursor protein, e.g., via enzymatic cleavage ofenzyme cleavage sites inserted adjacent to the N- and/or C-terminus ofthe immunogenic peptide, followed by suitable purification.

In view of the limited length of the immunogenic peptides of theinvention, they can be prepared by chemical peptide synthesis, whereinpeptides are prepared by coupling the different amino acids to eachother. Chemical synthesis is particularly suitable for the inclusion ofe.g. D-amino acids, amino acids with non-naturally occurring side chainsor natural amino acids with modified side chains such as methylatedcysteine. Chemical peptide synthesis methods are well described andpeptides can be ordered from companies such as Applied Biosystems andother companies. Peptide synthesis can be performed as either solidphase peptide synthesis (SPPS) or contrary to solution phase peptidesynthesis. The best-known SPPS methods are t-Boc and Fmoc solid phasechemistry which is amply known to the skilled person. In addition,peptides can be linked to each other to form longer peptides using aligation strategy (chemoselective coupling of two unprotected peptidefragments) as originally described by Kent (Schnolzer & Kent (1992) Int.J. Pept. Protein Res. 40, 180-193) and reviewed for example in Tam etal. (2001) Biopolymers 60, 194-205. This provides the tremendouspotential to achieve protein synthesis which is beyond the scope ofSPPS. Many proteins with the size of 100-300 residues have beensynthesised successfully by this method. Synthetic peptides havecontinued to play an ever-increasing crucial role in the research fieldsof biochemistry, pharmacology, neurobiology, enzymology and molecularbiology because of the enormous advances in the SPPS.

The physical and chemical properties of an immunogenic peptide ofinterest (e.g. solubility, stability) is examined to determine whetherthe peptide is/would be suitable for use in therapeutic compositions.Typically this is optimised by adjusting the sequence of the peptide.Optionally, the peptide can be modified after synthesis (chemicalmodifications e.g. adding/deleting functional groups) using techniquesknown in the art.

Accordingly, in yet a further aspect, the present invention providesmethods for generating viral vector antigen-specific cytotoxic T cells(Tregs or CD4+ regulatory T-cells) either in vivo or in vitro (ex vivo).In particular said T cells are cytotoxic towards any cell presenting aviral vector antigen and are obtainable as a cell population. Theinvention extends to such (populations of) viral vector antigen-specificcytotoxic Tregs obtainable by the herein described methods.

In particular embodiments, methods are provided which comprise theisolation of peripheral blood cells, the stimulation of the cellpopulation in vitro by contacting an immunogenic peptide according tothe invention with the isolated peripheral blood cells, and theexpansion of the stimulated cell population, more particularly in thepresence of IL-2. The methods according to the invention have theadvantage that higher numbers of Tregs are produced and that the Tregscan be generated which are specific for the viral vector antigenicprotein (by using a peptide comprising an antigen-specific epitope).Alternatively, viral vector protein-specific cytotoxic T cells may beobtained by incubation in the presence of APCs presenting a viral vectorprotein-specific immunogenic peptide according to the invention aftertransduction or transfection of the APCs with a genetic constructcapable of driving expression of such immunogenic peptide. Such APCs mayin fact themselves be administered to a subject in need to trigger invivo in said subject the induction of the beneficial subset of cytotoxicCD4+ T-cells.

In an alternative embodiment, the Tregs can be generated in vivo, i.e.by the administration of an immunogenic peptide provided herein to asubject, and collection of the Tregs generated in vivo.

The viral vector protein- or antigen-specific regulatory T cellsobtainable by the above methods are of particular interest for use inthe manufacture of a medicament for preventing or suppressing in arecipient of gene therapy or gene vaccination the immune response to aviral vector, i.e., for any of the above-described uses of theimmunogenic peptides of the invention, said peptides can be replaced bysaid viral vector protein- or antigen-specific Tregs. Both the use ofallogeneic and autogeneic cells is envisaged. Any method comprising theadministration of said viral vector protein- or antigen-specific Tregsto a subject in need (i.e., for preventing or suppressing immuneresponse(s) to a viral vector) is also known as adoptive cell therapy.Such therapy is of particular interest in case of treating acute viralvector protein-specific immune reactions and relapses of such reactions.Tregs are crucial in immunoregulation and have great therapeuticpotential. The efficacy of Treg-based immunotherapy depends on the Agspecificity of the regulatory T cells. Moreover, the use of Ag-specificTreg as opposed to polyclonal expanded Treg reduces the total number ofTreg necessary for therapy.

The present invention also relates to nucleic acid sequences encodingthe immunogenic peptides of the present invention and methods for theiruse, e.g., for recombinant expression or in gene therapy. In particular,said nucleic acid sequences are capable of expressing the immunogenicpeptides of the invention.

The immunogenic peptides of the invention may indeed be administered toa subject in need by using any suitable gene therapy method. In any useor method of the invention for the treatment and/or suppression ofimmune response(s) to a viral vector, immunisation with an immunogenicpeptide of the invention may be combined with adoptive cell transfer of(a population of) Tregs specific for said immunogenic peptide and/orwith gene therapy. When combined, said immunisation, adoptive celltransfer and gene therapy can be used concurrently, or sequentially inany possible combination.

In gene therapy, recombinant nucleic acid molecules encoding theimmunogenic peptides can be used as naked DNA or in liposomes or otherlipid systems for delivery to target cells. Other methods for the directtransfer of plasmid DNA into cells are well known to those skilled inthe art for use in human gene therapy and involve targeting the DNA toreceptors on cells by complexing the plasmid DNA to proteins. In itssimplest form, gene transfer can be performed by simply injecting minuteamounts of DNA into the nucleus of a cell, through a process ofmicroinjection. Once recombinant genes are introduced into a cell, theycan be recognised by the cells normal mechanisms for transcription andtranslation, and a gene product will be expressed. Other methods havealso been attempted for introducing DNA into larger numbers of cells.These methods include: transfection, wherein DNA is precipitated withcalcium phosphate and taken into cells by pinocytosis; electroporation,wherein cells are exposed to large voltage pulses to introduce holesinto the membrane); lipofection/liposome fusion, wherein DNA is packedinto lipophilic vesicles which fuse with a target cell; and particlebombardment using DNA bound to small projectiles. Another method forintroducing DNA into cells is to couple the DNA to chemically modifiedproteins. Adenovirus proteins are capable of destabilising endosomes andenhancing the uptake of DNA into cells. Mixing adenovirus to solutionscontaining DNA complexes, or the binding of DNA to polylysine covalentlyattached to adenovirus using protein crosslinking agents substantiallyimproves the uptake and expression of the recombinant gene.Adeno-associated virus vectors may also be used for gene delivery intovascular cells. As used herein, “gene transfer” means the process ofintroducing a foreign nucleic acid molecule into a cell, which iscommonly performed to enable the expression of a particular productencoded by the gene. The said product may include a protein,polypeptide, anti-sense DNA or RNA, or enzymatically active RNA. Genetransfer can be performed in cultured cells or by direct administrationinto mammals. In another embodiment, a vector comprising a nucleic acidmolecule sequence encoding an immunogenic peptide according to theinvention is provided. In particular embodiments, the vector isgenerated such that the nucleic acid molecule sequence is expressed onlyin a specific tissue. Methods of achieving tissue-specific geneexpression are well known in the art, e.g., by placing the sequenceencoding an immunogenic peptide of the invention under control of apromoter, which directs expression of the peptide specifically in one ormore tissue(s) or organ(s). Expression vectors derived from viruses suchas retroviruses, vaccinia virus, adenovirus, adeno-associated virus,herpes viruses, RNA viruses or bovine papilloma virus, may be used fordelivery of nucleotide sequences (e.g., cDNA) encoding peptides,homologues or derivatives thereof according to the invention into thetargeted tissues or cell population. Methods which are well known tothose skilled in the art can be used to construct recombinant viralvectors containing such coding sequences. Alternatively, engineeredcells containing a nucleic acid molecule coding for an immunogenicpeptide according to the invention may be used in gene therapy. Inparticular embodiments of the present invention wherein the immunogenicpeptide is delivered through gene transfer, this can be ensured as partof the gene therapy to which the patient in subjected. Accordingly theimmunogenic protein is delivered simultaneously with the viral vectoritself (which is expected to generate the immune reaction).

Where the administration of one or more peptides according to theinvention is ensured through gene transfer (i.e. the administration of anucleic acid which ensures expression of peptides according to theinvention in vivo upon administration), the appropriate dosage of thenucleic acid can be determined based on the amount of peptide expressedas a result of the introduced nucleic acid.

The medicament of the invention is usually, but not necessarily, a(pharmaceutical) formulation comprising as active ingredient at leastone of the immunogenic peptides of the invention, a (population of)Tregs specific for said immunogenic peptide or a gene therapeutic vectorcapable of expressing said immunogenic peptide. Apart from the activeingredient(s), such formulation will comprise at least one of a(pharmaceutically acceptable) diluent, carrier or adjuvant. Typically,pharmaceutically acceptable compounds (such as diluents, carriers andadjuvants) can be found in, e.g., a Pharmacopeia handbook (e.g. US-,European- or International Pharmacopeia). The medicament orpharmaceutical composition of the invention normally comprises a(prophylactically or therapeutically) effective amount of the activeingredient(s) wherein the effectiveness is relative to the condition ordisorder to be prevented or treated. In particular, the pharmaceuticalcompositions of the invention are vaccines for prophylactic ortherapeutic application.

The medicament or pharmaceutical composition of the invention may needto be administered to a subject in need as part of a prophylactic ortherapeutic regimen comprising multiple administrations of saidmedicament or composition. Said multiple administrations usual occursequentially and the time-interval between two administrations can varyand will be adjusted to the nature of the active ingredient and thenature of the condition to be prevented or treated. The amount of activeingredient given to a subject in need in a single administration canalso vary and will depend on factors such as the physical status of thesubject (e.g. weight, age), the status of the condition to be preventedor treated, and the experience of the treating doctor, physician ornurse.

The term “diluents” refers for instance to physiological salinesolutions. The term “adjuvant” usually refers to a pharmacological orimmunological agent that modifies (preferably increases) the effect ofother agents (e.g., drugs, vaccines) while having few if any directeffects when given by themselves. As one example of an adjuvantaluminium hydroxide (alum) is given, to which an immunogenic peptide ofthe invention can be adsorbed. Further, many other adjuvants are knownin the art and can be used provided they facilitate peptide presentationin MHC-class II presentation and T cell activation. The term“pharmaceutically acceptable carrier” means any material or substancewith which the active ingredient is formulated in order to facilitateits application or dissemination to the locus to be treated, forinstance by dissolving, dispersing or diffusing the said composition,and/or to facilitate its storage, transport or handling withoutimpairing its effectiveness. They include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents (forexample phenol, sorbic acid, chlorobutanol), isotonic agents (such assugars or sodium chloride) and the like. Additional ingredients may beincluded in order to control the duration of action of the activeingredient in the composition. The pharmaceutically acceptable carriermay be a solid or a liquid or a gas which has been compressed to form aliquid, i.e. the compositions of this invention can suitably be used asconcentrates, emulsions, solutions, granulates, dusts, sprays, aerosols,suspensions, ointments, creams, tablets, pellets or powders. Suitablepharmaceutical carriers for use in said pharmaceutical compositions andtheir formulation are well known to those skilled in the art, and thereis no particular restriction to their selection within the presentinvention. They may also include additives such as wetting agents,dispersing agents, stickers, adhesives, emulsifying agents, solvents,coatings, antibacterial and antifungal agents (for example phenol,sorbic acid, chlorobutanol), isotonic agents (such as sugars or sodiumchloride) and the like, provided the same are consistent withpharmaceutical practice, i.e. carriers and additives which do not createpermanent damage to mammals. The pharmaceutical compositions of thepresent invention may be prepared in any known manner, for instance byhomogeneously mixing, coating and/or grinding the active ingredients, ina one-step or multi-steps procedure, with the selected carrier materialand, where appropriate, the other additives such as surface-activeagents. They may also be prepared by micronisation, for instance in viewto obtain them in the form of microspheres usually having a diameter ofabout 1 to 10 μm, namely for the manufacture of microcapsules forcontrolled or sustained release of the active ingredients.

Immunogenic peptides, homologues or derivatives thereof according to theinvention (and their physiologically acceptable salts or pharmaceuticalcompositions all included in the term “active ingredients”) may beadministered by any route appropriate to the condition to be preventedor treated and appropriate for the compounds, here the immunogenicproteins to be administered. Possible routes include regional, systemic,oral (solid form or inhalation), rectal, nasal, topical (includingocular, buccal and sublingual), vaginal and parenteral (includingsubcutaneous, intramuscular, intravenous, intradermal, intraarterial,intrathecal and epidural). The preferred route of administration mayvary with for example the condition of the recipient or with thecondition to be prevented or treated.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as solution or a suspension in an aqueous liquid ora non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste. A tablet may be made bycompression or moulding, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing in asuitable machine the active ingredient in a free-flowing form such as apowder or granules, optionally mixed with a binder, lubricant, inertdiluent, preservative, surface active or dispersing agent. Mouldedtablets may be made by moulding in a suitable machine a mixture of thepowdered compound moistened with an inert liquid diluent. The tabletsmay optionally be coated or scored and may be formulated so as toprovide slow or controlled release of the active ingredient therein.

A further aspect of the invention relates to isolated immunogenicpeptides comprising a T-cell epitope from a viral vector protein and,adjacent to said T-cell epitope or separated from said T-cell epitope bya linker, a [CST]-(X)2-[CST] motif, more particularly a C-(X)2-[CST] or[CST]-(X)2-C motif. In particular embodiments, the viral vector proteinis a viral protein. In further particular embodiments the viral vectorprotein is a capsid protein.

Viral vectors for the purpose of gene therapy or gene vaccination arehighly amenable to modifications by means of recombinant nucleic acidtechnology. In view of the above, a skilled person will further easilyenvisage that the modification to the viral vector T-cell epitope asapplied in the immunogenic peptides and their uses according to theinvention can be introduced immediately in the viral vector itself. Assuch, vaccination with the immunogenic peptides comprising a viralvector T-cell epitope and a redox motif (and/or the corresponding genevaccination and/or the corresponding adoptive cell transfer) may becomeredundant as the same beneficial effects can be obtained with a modifiedviral vector. Hence, the invention further encompasses modified viralvectors defined as isolated viral vectors characterised in that at leastone T-cell epitope present in at least one of the viral vector proteinsis modified by insertion in said viral vector protein, adjacent to saidT-cell epitope or separated from said T-cell epitope by a linker, of aC-(X)2-[CST] or [CST]-(X)2-C motif motif. More particularly the T-cellepitope is separated from the motif by a linker of at most 7 aminoacids. In particular embodiments, isolated viral vectors are providedcomprising at least one viral vector protein comprising a T cell epitopeand adjacent thereto or separated from said T-cell epitope by a linker,a C-(X)2-[CST] or [CST]-(X)2-C motif, wherein the motif does notnaturally occur within the viral vector protein within a sequence of 11amino acids N- or C-terminally adjacent to the T-cell epitope.

In particular embodiments thereof, said viral vector is furthercharacterised in that said modified T-cell epitope is capable of beingpresented by an MHC class II determinant. In another embodiment, saidisolated viral vectors are further characterised in that their celltransducing properties are not significantly altered compared to thesame viral vector not carrying the T-cell epitope modification.

The present invention will now be illustrated by means of the followingexamples, which are provided without any limiting intention.Furthermore, all references described herein are explicitly includedherein by reference.

EXAMPLES Example 1 In Vitro Elicitation of Cytotoxic Regulatory T Cellsfrom Memory T Cells Specific for Viral Vector Protein

Adenovirus of serotype 5 (Ad.RR5, E1/E3-deleted) is a commonly usedserotype for gene therapy and for gene vaccination. Both in man andmouse a strong immune response is induced towards the major capsidprotein hexon. This is used as a model to determine whether cytotoxicregulatory T cells could be derived from cells obtained by immunisingmice with the virus vector.

Thus, 5 μL of a solution containing 2×10¹¹ viral particles/ml isadministered by the intravenous route to 6 weeks old C57BI/6 mice. Tendays later, the spleen of such mice is recovered and CD4+ T cellspurified by magnetic sorting.

A T cell epitope was identified within the sequence of the major capsidprotein, by a combination of algorithms. A T cell epitope encompassingamino acid residues 912-921 was selected, with sequence: PTLLYVLFEV (SEQID NO:12; natural epitope). A synthetic peptide encoding this naturalepitope sequence was obtained. A second peptide additionally containinga thioreductase consensus sequence (or redox motif) was synthesised andhas the sequence: CHGCPTLLYVLFEV (SEQ ID NO:13; redox motif underlined;modified epitope).

CD4+ T cells obtained from Ad.RR5-immunized mice are cultured in thepresence of spleen adherent cells used as antigen-presenting cellspreincubated with either peptide of SEQ ID NO:12 or peptide of SEQ IDNO:13.

After several cycles of restimulation, the CD4+ T cells are cloned bylimiting dilution and allowed to rest for 10 days. Clones expanded withthe peptide of SEQ ID NO:12 are then compared to clones expanded withthe peptide of SEQ ID NO:13 in an assay in which purified B cells fromnaïve C57BI/6 mice are used as antigen-presenting cells. Said B cellsare loaded with peptides of SEQ ID NO:12 or 13 and cultured in thepresence of the cloned CD4+ T cells. After an incubation of 18 h, theinduction of apoptosis in B cells is measured by binding of annexin Vand Facs analysis.

These experiments demonstrate that is possible to induce CD4+ T cellsfrom memory T cells specific for viral vector protein by using viralvector T cell epitopes modified to comprise a thioreductase consensussequence (redox motif) wherein the induced CD4+ T cells are capable ofeliciting apoptosis of cells presenting either the natural or modifiedviral vector T cell epitope.

Example 2 Preimmunisation with a Viral Vector T Cell Epitope Containinga Thioreductase Consensus Sequence Prevents Subsequent Development ofCD4+ Effector T Cells Towards Viral Vector Proteins

Preimmunisation (prior to any contact with viral vector protein) isperformed with peptides of SEQ ID NO:13 (see Example 1) in order toprevent the development of a CD4+ effector T cells directed against theadenovirus vector proteins.

To this end, a group C57BI/6 mice are immunised with 25 μg of peptide ofSEQ ID NO:13 in CFA (complete Freund's adjuvant; first immunisation) orIFA (incomplete Freund's adjuvant; second and third immunisation).Injections are made subcutaneously at fortnight intervals. A controlgroup of mice receives immunisation with peptide of SEQ ID NO:12 and athird group is immunised with a sham peptide. All mice then receive anintravenous injection containing 10⁹ adenoviral particles (Ad.RR5, seeExample 1). Ten days later, the spleen is recovered and CD4+ T cellspurified by sorting on magnetic beads.

The capacity of CD4+ T cells to proliferate in the presence of peptideof SEQ ID NO:12 presented by B cells of naïve animals is measured. Thesame experiment is repeated with the full adenovirus vector instead ofthe peptide of SEQ ID NO:12.

This experiment indicates that preimmunisation with a viral vector Tcell epitope modified to contain a thioreductase consensus sequence(redox motif) is able to prevent subsequent development of CD4+ effectorT cells towards the natural T cell epitope (unmodified, not containingthe redox motif) presented as peptide or presented comprised in a fullvector protein.

Example 3 Suppression of Pre-existing CD4+ Effector Cells to ViralVector Proteins by Immunisation with a T Cell Epitope Modified toComprise a Thioreductase Consensus Sequence

The experiment as described in Example 2 is repeated in a setting inwhich all mice first received an intravenous injection of Ad.RR5 (seeExample 1). Ten days later, groups of mice are immunised as above withthe peptide of SEQ ID NO:13 (T cell epitope modified to comprise a redoxmotif; see Example 1), the peptide of SEQ ID NO:12 (natural T cellepitope, unmodified; see Example 1), or with a sham peptide.

Two weeks after the last immunisation with the peptides, spleens arerecovered, CD4+ T cells prepared as above and the capacity of CD4+ Tcells to proliferate is measured in the presence of adenoviral proteinspresented by B cells.

This experiment indicates that immunisation with a viral vector T cellepitope modified to comprise a thioreductase consensus sequence (redoxmotif) can suppress a CD4+ effector T cell response already existing dueto prior immunisation with viral vector proteins.

Example 4 Killing of Splenic B Cells with a T Cell Line Specific forHAdV-5

TCL lines were obtained from mice immunised with natural sequence555-563 (SEQ ID NO:14; YVPFHIQVP) from Late Protein 2 derived from humanadenovirus 5 (HAdV-5) (wt TCL) or with the same sequence but modified byaddition of a thioreductase motif (underlined) separated from the firstMHC class II anchoring residue by two Gly residues (SEQ ID NO:15;CGPCGGYVPFHIQVP) (cc TCL). Splenic B cells were stained with afluorescent membrane marker, loaded with peptide of SEQ ID NO:14 andco-cultured with each of the two T cell lines separately. Cell mortalitywithin the B cell population was analysed after 20 hours on a flowcytometer. Mortality was deducted from size exclusion dot-plots(baseline mortality (22%) was subtracted from test samples). Results aredepicted in FIG. 1 and illustrate that an immune response to theadenovirus can be eliminated by using a peptide according to theinvention, thus proving the validity of this technology as a means tocounter immune responses to viral vectors as used in gene therapy andgene vaccination.

I claim:
 1. An isolated immunogenic peptide comprising: an MHC class IIT-cell epitope from a viral protein from a viral vector for gene therapyor for gene vaccination, and a C-(X)2-[CST] or [CST]-(X)2-Cthioreductase motif, wherein said motif is immediately adjacent to saidT-cell epitope or is separated from said T- cell epitope by a linker,wherein said C-(X)2-[CST] or [CST]-(X)2-C thioreductase motif does notnaturally occur within a region of 11 amino acids N- or C-terminallyadjacent to said T-cell epitope in said viral vector protein.
 2. Thepeptide according to claim 1, wherein said linker consists of at most 7amino acids.
 3. The peptide according to claim 1, wherein said linkerconsists of at most 4 amino acids.
 4. The peptide according to claim 1,which has a length of between 12 and 50 amino acids.
 5. The peptideaccording to claim 1, which has a length of between 12 and 30 aminoacids.
 6. The peptide according to claim 1, wherein said motif isC-(X)2-C.
 7. The peptide according to claim 1, wherein said immunogenicpeptide further comprises an endosomal targeting sequence.
 8. Thepeptide according to claim 1, wherein said C-(X)2-[CST] or [CST]-(X)2-Cmotif is positioned N-terminally of the T-cell epitope.
 9. The peptideaccording to claim 1, wherein at least one X in said C-(X)2-[CST] or[CST]-(X)2-C motif is Gly, Ala, Ser or Thr.
 10. The peptide according toclaim 1, wherein at least one X in said C-(X)2-[CST] or [CST]-(X)2-Cmotif is His or Pro.
 11. The peptide according to claim 1 wherein atleast one C in said C-(X)2-[CST] or [CST]-(X)2-C motif is methylated.12. A method of producing the isolated immunogenic peptide of claim 1comprising the steps of: identifying an MHC class II T cell epitope in aprotein sequence of a viral protein of a viral vector for gene therapyor for gene vaccination, and producing a peptide comprising saididentified T cell epitope and a sequence with a C-(X)2-[CST] or[CST]-(X)2-C thioreductase motif, wherein said T cell epitope and saidthioreductase motif are immediately adjacent or separated by a linker,wherein said C-(X)2-[CST] or [CST]-(X)2-C thioreductase motif does notnaturally occur within a region of 11 amino acids N- or C-terminallyadjacent to said T-cell epitope in said viral vector protein.
 13. Amethod for obtaining a population of viral vector proteinantigen-specific cytotoxic CD4+ T cells which induce apoptosis of APC(Antigen Presenting Cells) presenting an MHC class II T cell epitope ofsaid viral vector protein, the method comprising the steps of: providingperipheral blood cells; contacting said cells in vitro with the isolatedimmunogenic peptide of claim 1; and expanding said cells in the presenceof Interleukin 2 (IL-2).
 14. A method for obtaining a population ofviral vector protein antigen-specific cytotoxic CD4+ T cells whichinduce apoptosis of APC presenting a MHC class II T cell epitope of saidviral protein from a vector for gene therapy or for gene vaccination,the method comprising the steps of: providing the isolated immunogenicpeptide of claim 1; administering said immunogenic peptide to a subject;and obtaining said population of viral vector antigen-specific CD4+ Tcells from said subject.
 15. A method of preventing or suppressing in arecipient of gene therapy vector or gene vaccination vector the immuneresponses to a viral vector, the method comprising the step ofadministering a population of antigen-specific cytotoxic CD4+ T cellswhich induce apoptosis of APC presenting the isolated immunogenicpeptide of claim 1.